Aberrant AKT activation drives well-differentiated
liposarcoma
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Citation
Gutierrez, A. et al. “Aberrant AKT Activation Drives Welldifferentiated Liposarcoma.” Proceedings of the National
Academy of Sciences 108.39 (2011): 16386–16391. Web. 13
Apr. 2012.
As Published
http://dx.doi.org/10.1073/pnas.1106127108
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Proceedings of the National Academy of Sciences (PNAS)
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Final published version
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Wed May 25 16:05:16 EDT 2016
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http://hdl.handle.net/1721.1/70023
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Detailed Terms
Aberrant AKT activation drives
well-differentiated liposarcoma
Alejandro Gutierreza,b,1, Eric L. Snyderc,d,e,f, Adrian Marino-Enriquezc, Yi-Xiang Zhangf,g, Stefano Sioleticf,g,
Elena Kozakewicha, Ruta Grebliunaitea, Wen-bin Ouc, Ewa Sicinskad,f, Chandrajit P. Rautg,h,
George D. Demetrif,g, Antonio R. Perez-Ataydei, Andrew J. Wagnerf,g, Jonathan A. Fletcherc,g,
Christopher D. M. Fletcherc, and A. Thomas Looka,b,1
a
Department of Pediatric Oncology, dDepartment of Medical Oncology and Center for Molecular Oncologic Pathology, fThe Ludwig Center at Dana-Farber/
Harvard Cancer Center, and gCenter for Sarcoma and Bone Oncology, The Dana-Farber Cancer Institute, MA 02215; bDivision of Hematology/Oncology,
i
Department of Pathology, Children’s Hospital, Boston, MA 02115; cDepartment of Pathology and hDepartment of Surgery, Brigham and Women’s Hospital,
Boston, MA 02115; and eThe Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139
Edited* by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved August 15, 2011 (received for review April 21, 2011)
Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its
pathobiology and development of effective targeted therapies.
Here, we show that zebrafish expressing constitutively active Akt2
in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53
wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53homozygous mutant zebrafish (P = 0.013), indicating that aberrant
Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of
the closely related dedifferentiated liposarcoma (DDLPS) subtype,
revealed immunohistochemical evidence of AKT activation in 27%
of cases. Western blot analysis of a panel of cell lines derived from
patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured
in serum-free media. Moreover, BEZ235, a small molecule inhibitor
of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar
concentrations. Our findings are unique in providing an animal
model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this
chemoresistant sarcoma.
L
iposarcoma is the most common sarcoma of humans, affecting ∼2,000 individuals per year in the United States (1).
These tumors are classified into five histopathologic subtypes,
with well-differentiated liposarcoma (WDLPS) accounting for
∼50% of cases, and dedifferentiated liposarcoma (DDLPS), a
closely related subtype that appears to arise from further malignant progression of WDLPS, accounting for an additional 9%
to 18% of cases (1–3). Liposarcomas are generally thought to
arise de novo rather than from preexisting benign lesions, and
most patients lack recognized causative factors. Although complete surgical resection can be curative, WDLPS often develops
in deep anatomic locations, such as the retroperitoneum or
mediastinum, where its propensity to enwrap vital structures
typically makes complete surgical resection difficult or impossible, leading to high morbidity and mortality rates (1, 4). Radiation and chemotherapy have limited efficacy in the treatment of
WDLPS (5, 6). Indeed, there are no systemic therapeutic regimens known to improve survival when complete surgical resection is not feasible, underscoring the need for an improved
molecular understanding of WDLPS to stimulate the development of effective targeted therapies.
The MDM2-p53 pathway plays a prominent role in WDLPS
pathogenesis, with the vast majority of human tumors harboring
either MDM2 amplifications or p53 mutations (6–10). Moreover,
individuals with germ-line p53 mutations appear to be at increased risk of WDLPS development at a very young age (11).
16386–16391 | PNAS | September 27, 2011 | vol. 108 | no. 39
Regions of chromosome 12q13-15 are often amplified in welldifferentiated and dedifferentiated liposarcomas, typically involving MDM2, CDK4, and HMGA2, along with several other
genes (6, 10, 12, 13); JUN can also be amplified in WDLPS cases
that have a dedifferentiated component (14). Further dissection
of WDLPS molecular pathogenesis has been greatly impeded by
the lack of animal models suitable for experimental analysis.
Oncogenic signal transduction through the PI3K-AKT pathway, which is widely dysregulated in human cancer, is normally
down-regulated by the PTEN tumor suppressor (15). Individuals
with germ-line PTEN-inactivating mutations frequently develop
multiple lipomas (benign adipocytic neoplasms) (16), and AKT
activation has been described in human liposarcomas (17), suggesting that the PI3K-AKT pathway is involved in adipocyte
transformation. Here we show that expression of constitutively
active Akt2 in zebrafish mesenchymal progenitors induces
WDLPS, thus being unique in providing an animal model for
future investigation of this disease. Moreover, we also show that
AKT pathway inhibition impairs viability in human cell lines
derived from patients with WDLPS and DDLPS, thus implicating AKT as a previously unexplored therapeutic target in these
chemoresistant sarcomas.
Results
Expression of Constitutively Active Akt2 Induces Well-Differentiated
Liposarcoma. To test the hypothesis that Akt is a WDLPS onco-
gene that collaborates with p53 inactivation during adipocyte
transformation, we in-crossed zebrafish harboring heterozygous
p53M214K mutations, which encode a transactivation-defective
p53 protein (18), and all resultant embryos were microinjected at
the one-cell stage with a rag2:myr-mAkt2 expression construct
(Fig. 1A). This construct encodes a myristoylated, constitutively
active mouse Akt2 transgene (19) driven by a zebrafish rag2
Author contributions: A.G., E.L.S., A.M.-E., W.-b.O., J.A.F., C.M.D.F., and A.T.L. designed
research; A.G., E.L.S., A.M.-E., Y.-X.Z., S.S., E.K., R.G., and W.-b.O. performed research;
E.L.S., S.S., E.S., C.P.R., G.D.D., A.J.W., J.A.F., and C.M.D.F. contributed new reagents/analytic tools; A.G., E.L.S., A.M.-E., Y.-X.Z., S.S., E.K., W.-b.O., C.P.R., G.D.D., A.R.P.-A., J.A.F.,
C.M.D.F., and A.T.L. analyzed data; and A.G. and A.T.L. wrote the paper.
Conflict of interest statement: C.P.R. is a consultant for Novartis and participates in clinical
trials of Novartis. G.D.D. is a consultant for Novartis, Pfizer, Ariad, Johnson & Johnson,
PharmaMar, Genentech, Infinity Pharmaceuticals, EMD-Serono, Glaxo Smith Kline, Amgen, Daiichi-Sankyo, ArQule, Enzon, Millenium/Takeda; is a member of the scientific
advisory board of Plexxikon, ZioPharm, Nereus, N-of-One, and Kolltan Pharmaceuticals;
and participates in clinical trials of Novartis, Pfizer, Ariad, Johnson & Johnson, PharmaMar, and Infinity Pharmaceuticals. A.J.W. is a consultant for Novartis, Roche/Genentech,
Sanofi, Pfizer, EMD-Serono, and participates in clinical trials supported by Novartis,
Roche/Genentech, Pfizer, and Exelixis.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence may be addressed. E-mail: thomas_look@dfci.harvard.edu or
Alejandro_Gutierrez@dfci.harvard.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1106127108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1106127108
promoter fragment that drives ectopic expression in mesenchymal progenitors (20). Zebrafish injected with rag2:myr-mAkt2
developed externally visible solid tumors between 1 and 4 mo of
age; the tumor incidence rates were 29% in p53-homozygous
mutants, 6% in p53 heterozygotes, and 8% in their p53 wild-type
siblings (P = 0.01) (Fig. 1 B and C). Histologic analyses revealed
that 91% of these tumors consisted of locally invasive masses of
adipocytes showing considerable variation in cell size, together
with scattered atypical stromal cells with hyperchromatic nuclei
and lipoblasts characterized by multivacuolated cytoplasm and
large hyperchromatic pleomorphic nuclei, findings that are diagnostic of WDLPS in humans (Fig. 1 D–F) (2), whereas the
remaining 9% of tumors were osteosarcomas, as described below. Immunohistochemical analysis revealed strong reactivity for
phospho-AKT(Ser473) in the tumor cells of rag2:myr-mAkt2
transgenic zebrafish but not in the normal fat of control rag2:
GFP transgenic fish, indicating expression of the constitutively
active Akt2 transgene (Fig. 1 G–I). Moreover, none of the control p53-homozygous mutant zebrafish injected with rag2:GFP
(n = 60) developed tumors by 6 mo of age.
In addition, one p53-homozygous mutant injected with rag2:
myr-mAkt2 developed a rigid mass at the base of the dorsal fin
wt/mut
tion is also involved in human liposarcoma pathogenesis, we
performed immunohistochemical analysis for phospho-AKT
(Ser473) on clinical specimens from 58 patients with well-differentiated and dedifferentiated liposarcomas. These studies
demonstrated AKT activation in a subset of the human WDLPS
or DDLPS cases (Fig. 3 A–F), including 22% of the pure
WDLPS (n = 23) and 46% of pure DDLPS (n = 13) tumors
analyzed (Fig. 3J). We also included human tumors containing
both well-differentiated and dedifferentiated liposarcoma components in our analysis (n = 22), which revealed phospho-AKT
positivity in 32% of the well-differentiated components and 45%
in the dedifferentiated components of these cases (Fig. 3J).
Given that phospho-AKT is a labile epitope in clinical speci-
B
X
tp53
Aberrant AKT Pathway Activation in Primary Human Well-Differentiated
and Dedifferentiated Liposarcomas. To test whether AKT activa-
tp53
wt/mut
Inject:
rag2 promoter- myr
Assess Tumor Onset
mAkt2
Genotype
Solid Tumor Incidence (%)
A
that demonstrated increased lobulation and vascularity compared with the WDLPS tumors (Fig. 2 A and B). Histologic
analysis revealed that this mass consisted predominantly of osteoid interspersed with large malignant cells characterized by
pleomorphic nuclei, features that are diagnostic of osteosarcoma
in humans (Fig. 2 C and D) (2).
P = 0.013
30
p53 mut/mut (n=21)
p53 mut/wt (n=52)
p53 wt/wt (n=25)
20
10
0
0
2
4
6
Age (months)
8
C
Control
Well-Differentiated Liposarcoma
E
F
G
H
I
Fig. 1. Constitutive Akt activation drives WDLPS in the zebrafish. (A) Experimental design. (B) Solid tumor incidence in p53 wild-type, heterozygous, or p53M214K
homozygous mutant siblings injected with a rag2:myr-mAkt2 transgene at the one-cell stage. P value calculated via log-rank test. (C) Representative rag2:myrmAkt2-injected zebrafish, which developed what appear to be two independent solid tumors. The animal shown was p53-homozygous mutant. (Scale bar, 5
mm.) Note that the image shown in (C) consists of merged adjacent photomicrographs. (D) Control H&E-stained zebrafish section. Arrow points to normal
subcutaneous adipocytes. (E) Low-magnification view of an H&E section through the zebrafish shown in C, demonstrating a locally invasive mass consisting of
well-differentiated adipocytes with significant variation in cell size. (F) High-magnification view of H&E section demonstrating a representative lipoblast
scattered throughout these tumors, characterized by a multivacuolated cytoplasm and large hyperchromatic pleomorphic nuclei. (G) Phospho-AKT immunohistochemistry on a control zebrafish section. Arrow points to normal subcutaneous adipocytes, which lacked detectable pAKT staining. (H and I) Phospho-AKT
immunohistochemistry on tumor sections from the zebrafish shown in C, revealing strong immunoreactivity for phospho-AKT in tumor cells of rag2:myr-mAkt2transgenic zebrafish. (Scale bars, 100 μm.)
Gutierrez et al.
PNAS | September 27, 2011 | vol. 108 | no. 39 | 16387
MEDICAL SCIENCES
phospho-AKT
H&E
D
A
demonstrating evidence of S6 activation (Fig. 3K). We found no
immunohistochemical evidence of AKT or S6 phosphorylation in
lipomas or in normal adipose tissue (Fig. 3 D and G, and Fig. S1).
To determine whether AKT activation is aberrant in human
WDLPS and DDLPS, we took advantage of a panel of cell lines
derived from patients with these sarcomas to evaluate phosphorylation of AKT and of its downstream target GSK3β after
4 h of growth in serum-free conditions. Strikingly, Western blot
analysis revealed persistent phosphorylation of AKT and of its
downstream target GSK3β in all eight cell lines examined, even
under such serum-starved conditions. In contrast, serum starvation resulted in silencing of AKT and GSK3β phosphorylation in
control SU-CCS-1 clear cell sarcoma cells (Fig. 4).
B
C
D
Fig. 2. Osteosarcoma development in a rag2:myr-mAkt2-injected, p53-homozygous mutant zebrafish. (A and B) A p53-homozygous mutant zebrafish
injected with the rag2:myr-mAkt2 expression construct developed a solid
lobulated mass at the base of the dorsal fin. Note that the image shown in A
consists of merged adjacent photomicrographs. (Scale bars, 1 mm.) (C and D)
H&E-stained sections at low and high magnification, respectively, demonstrate a mass consisting predominantly of osteoid matrix interspersed with
large malignant cells with pleomorphic nuclei, features that in humans are
diagnostic of osteoblastic osteosarcoma. (Scale bars, 100 μm.)
mens, we also performed immunohistochemistry for phospho-S6
(Ser235/236), a downstream target of the AKT pathway (21).
Similar results (Fig. 3 G–I) were obtained, with 17% of pure
WDLPS (n = 23), 41% of well-differentiated WDLPS/DDLPS
components (n = 22), and 47% of DDLPS (pure DDLPS, n =
13; dedifferentiated WDLPS/DDLPS components, n = 21),
Lipoma
WDLPS
PI3K-AKT-Mammalian Target of Rapamycin Pathway Inhibition Impairs the Viability of Human Liposarcoma Cells. To determine
whether human WDLPS and DDLPS cells are dependent on
aberrant AKT pathway activation, we treated four cell lines (two
WDLPS and two DDLPS) with BEZ235, a dual-specificity inhibitor of PI3K and both mammalian target of rapamycin
(mTOR) complexes (22) that effectively silences AKT pathway
activation in these cells (Fig. 5A). BEZ235 treatment for 72 h
decreased the viability of all cell lines tested, with IC50 values
ranging from 13 to 75 nM (Fig. 5B). Treatment with rapamycin,
an mTORC1 inhibitor, had less of an effect on viability (Fig. 5C),
suggesting that the PI3K-AKT pathway plays both mTORC1dependent and -independent roles in the pathobiology of
WDLPS and DDLPS. Analysis of cell-cycle profiles revealed that
BEZ235 treatment induced G1 arrest at nanomolar concentrations (Fig. 5D), whereas apoptosis was induced only at a 1-μM
concentration (Fig. 5E).
DDLPS
B
C
D
E
F
G
H
I
100
Negative
Intermediate
High
80
60
40
20
K
pS6 IHC Staining (%)
J
pAKT IHC Staining (%)
pS6
pAKT
H&E
A
Negative
Intermediate
High
100
80
60
40
20
0
0
Pure
WDLPS
(n=23)
Well-Diff
(n=22)
Dediff
(n=22)
Pure
DDLPS
(n=13)
Cases with WDLPS &
DDLPS components
16388 | www.pnas.org/cgi/doi/10.1073/pnas.1106127108
Pure
WDLPS
(n=23)
Well-Diff
(n=22)
Dediff
(n=21)
Cases with WDLPS &
DDLPS components
Pure
DDLPS
(n=13)
Fig. 3. AKT pathway activation in primary human well-differentiated and dedifferentiated liposarcomas. (A–C) H&E
staining of human lipoma, WDLPS, and DDLPS specimens. (D–F)
Immunohistochemistry for Ser473-phosphorylated AKT in a
representative lipoma and AKT-positive WDLPS and DDLPS
clinical specimens. (Scale bar, 100 μm.) (G–I) Immunohistochemistry for phospho-S6 ribosomal protein (Ser235/236) in
a representative lipoma, as well as AKT-positive WDLPS and
DDLPS clinical specimens. (J and K) Quantitation of phosphoAKT and phospho-S6 immunohistochemistry in pure WDLPS,
pure DDLPS, and in human tumors with mixed well-differentiated and dedifferentiated liposarcoma components.
Gutierrez et al.
1
2
3
4
5
6
A
7
Controls
+
8
+
p-GSK3β
GSK3β
GSK3β
vinculin
vinculin
B
Relative Cell Number
p-GSK3β
Gutierrez et al.
0
0
1
3
10
C
1.2
30 100 300 1000
AKT
AKT
1
2
6
LP6
1.2
1.0
0.8
0.6
0.4
0.2
0
0
0.1
1
10
100
1
2
6
LP6
1.0
0.8
0.6
0.4
0.2
0
1000
0
0.1
BEZ235 (nM)
D
1
10
100 1000
Rapamycin (nM)
G2/M
S
G0/G1
E
Early Apoptosis
% of Cells
80
60
40
20
0
Late Apoptosis
15
100
% of Cells
Discussion
We have demonstrated that expression of activated Akt2 in mesenchymal progenitors drives WDLPS in transgenic zebrafish, and
that nearly one-third of clinical specimens from primary cases of
human WDLPS and DDLPS showed immunohistochemical evidence of AKT pathway activation. Moreover, treatment with the
PI3K-AKT pathway inhibitor BEZ235 inhibited viability in all cell
lines derived from patients with WDLPS and DDLPS that we
tested. Taken together, our findings implicate a central role for
oncogenic AKT signaling in the molecular pathogenesis of human
WDLPS and DDLPS, and suggest the need for clinical trials of
AKT pathway inhibitors in patients with unresectable disease, for
whom there are currently no known effective therapies.
Our analyses of primary human tumors revealed an increased
frequency and intensity of staining for phospho-AKT and phosphoS6 ribosomal protein in dedifferentiated liposarcomas compared with their well-differentiated counterparts. These findings
suggest the intriguing possibility that AKT activation may define
a subset of WDLPS, which is particularly prone to dedifferentiation, a process that may be caused in part by the acquisition
of additional oncogenic abnormalities further potentiating signaling through the AKT pathway. However, we cannot rule out
the possibility that this apparent difference may simply be related
to the greater difficulty of detecting phosphorylated epitopes in
WDLPS sections, in which most of the tumor mass consists of
large fat vacuoles within malignant yet well-differentiated adipocytes, whereas the densely cellular DDLPS tumors have a much
greater number of cellular elements in which AKT phosphorylation can be assessed per section. Further studies will be required
to establish the mechanisms underlying this observation.
Recent work has revealed that 18% of myxoid/round-cell liposarcomas harbor activating mutations in PIK3CA, encoding the
catalytic subunit of class IA PI3K (13). Myxoid/round-cell liposarcomas are characterized by t(12;16)(q13-14;p11) translocations, resulting in expression of TLS-CHOP fusion proteins,
whereas these tumors lack the 12q amplifications characteristic
of WDLPS/DDLPS, leading most investigators to believe that
these liposarcoma subtypes are biologically distinct (1, 2). S6K1,
a direct target of mTORC1 downstream of PI3K-AKT, has recently been shown to be required for the earliest stages of adipogenesis (23), providing one plausible mechanism to explain
selection for AKT pathway activation in diverse liposarcoma
subtypes. Nevertheless, the fact that expression of activated AKT
in p53-mutant zebrafish drives development of WDLPS but not
myxoid/round-cell liposarcomas indicates that AKT activation
and p53 mutation can be early events in WDLPS pathogenesis,
whereas expression of the TLS-CHOP fusion may be required in
addition to PI3K-AKT pathway activation in the genesis of
myxoid/round-cell liposarcomas.
-
p-AKT
p-AKT
Fig. 4. Aberrant AKT activation in human cell lines derived from patients
with WDLPS and DDLPS. Western blot analysis for phosphorylation of AKT
and its downstream target GSK3β was performed in a panel of cell lines
derived from patients with WDLPS (samples 1–3) or DDLPS (samples 4–8),
grown in serum-free conditions for 4 h before analysis. Control is the SU-SSC-1
clear-cell sarcoma cell line, shown in the presence and absence of serum.
Sample 1 was run on both Western blots as a control for interblot variability.
LP6 BEZ235 (nM)
10
5
0
0
1
3
10
30 100 300 1000
BEZ235 (nM)
0
30
100
300
1000
BEZ235 (nM)
Fig. 5. PI3K-AKT-mTOR pathway inhibitors impair viability in human WDLPS
and DDLPS cells. (A) Western blot analysis of the LP6 cell line, derived from
a patient with DDLPS, demonstrating that BEZ235 effectively inhibits phosphorylation of AKT and its downstream target GSK3β at nanomolar concentrations. Positive and negative controls are the SU-SSC-1 cell line grown in the
presence and absence of serum, respectively. (B) Viability of four cell lines
derived from patients with WDLPS (cells 1 and 2) or DDLPS (cells 6 and LP6) was
assessed after 72 h of BEZ235 treatment using the CellTiter-Glo luminescent
cell viability assay. Values represent mean ± SEM (n = 3 replicates). IC50 values
were 19, 32, 14, and 75 nM, respectively, for cells 1, 2, 6, and LP6. (C) Viability of
WDLPS/DDLPS cell lines after 72 h of rapamycin treatment. (D) Effect of BEZ235
treatment on cell cycle distribution of LP6 cells examined by flow cytometry
analysis at 24 h. Data shown are representative of two independent experiments. (E) Effect of BEZ235 treatment on apoptosis of LP6 cells, assessed by
Annexin V and 7-AAD double-staining. Data shown are representative of two
independent experiments.
Current knowledge of the molecular pathogenesis of WDLPS
has been driven by genetic analyses of human tumors, which have
revealed that almost all cases harbor MDM2 amplification or TP53
mutations (7–10). Evidence also suggests that individuals with
germ-line TP53 mutations are at increased risk of WDLPS development at a very young age (11). By demonstrating that activated Akt2 and p53 mutations collaborate in the zebrafish, we have
now experimentally demonstrated the long-suspected role of p53
as a tumor suppressor in WDLPS. These tumors are also characterized by recurrent amplifications of distinct regions of chromosome 12q13-15 (6, 10, 12), but until now it has not been possible to
identify which of the involved genes are WDLPS oncogenes
driving the selection for these amplifications, and which are merely
nonpathogenic “passengers.” Furthermore, although dedifferentiated liposarcoma is thought to arise because of further malignant transformation of WDLPS, it has previously been impossible to directly test the ability of candidate genetic lesions to
drive this transformation in a physiological context. The zebrafish
model we describe now provides a platform for experimental
PNAS | September 27, 2011 | vol. 108 | no. 39 | 16389
MEDICAL SCIENCES
1
Serum
DDLPS
Relative Cell Number
WDLPS
Control
studies to dissect molecular pathogenesis and discover novel
therapeutic targets in this chemoresistant tumor.
Inhibitors. BEZ235 and rapamycin were purchased from AXON Medchem and
Calbiochem, respectively.
Materials and Methods
Western Blotting. Whole-cell lysates were prepared using lysis buffer (1%
Nonidet P-40, 50 mM Tris-HCl pH 8.0, 100 mM sodium fluoride, 30 mM sodium
pyrophosphate, 2 mM sodium molybdate, 5 mM EDTA, and 2 mM sodium
orthovanadate) containing protease inhibitors (10 μg/mL aprotinin, 10 μg/mL
leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The lysates were
then rocked overnight at 4 °C. Lysates were cleared by centrifugation at
16,100 × g for 30 min at 4 °C, and protein concentrations were determined
with a Bio-Rad protein assay (Bio-Rad Laboratories). Equal amounts of protein were separated by SDS/PAGE, blotted to nitrocellulose membranes
(Schleicher and Schuell) and then stained with the following antibodies: AKT
(Cell Signaling Technology; #9272; 1:500), phospho-AKT(Ser473) (Cell Signaling
Technology; #9271; 1:500), phospho-GSK3β(Ser9) (Cell Signaling Technology;
#9336; 1:1,000), GSK3 (Santa Cruz; #sc-7291; 1:500), and vinculin (SigmaAldrich; #V4505; 1:500). The hybridization signals were detected by chemiluminescence (ECL, Amersham Biosciences) and captured using a LAS1000-plus
chemiluminescence imaging system (Fujifilm).
Zebrafish Husbandry, Mutant Lines, and Imaging. Zebrafish husbandry was
performed as previously described (24), in accord with protocols approved by
the Dana-Farber Cancer Institute Animal Care and Use Committee. The
p53M214K-mutant zebrafish line was previously described (18). Zebrafish
images were obtained using a Nikon SMZ1500 microscope, Nikon DS2MBWc
camera, and NIS-Elements F Package Ver. 3.00 (Nikon Instruments Inc.).
Expression Constructs. The rag2:myr-mAkt2 construct was generated by
placing a myristoylated murine Akt2 transgene (19), which is constitutively
activated as a result of constitutive membrane localization, downstream of
a zebrafish rag2 promoter fragment (25) in a modified pBluescript vector,
wherein the rag2:myr-mAkt2 construct is flanked by recognition sequences
for I-SceI meganuclease.
Generation of Transgenic Zebrafish. Circular rag2:myr-mAkt2 plasmid DNA
(30 pg) was microinjected along with I-SceI meganuclease (New England
Biolabs) into one-cell stage zebrafish embryos from the AB wild-type strain,
as previously described (26).
Zebrafish Paraffin Embedding and Sectioning. Zebrafish were killed in tricaine
anesthetic, fixed in 4% paraformaldehyde at 4 °C for 2 d, decalcified with
0.25 M EDTA (pH 8.0) for 2 d, dehydrated in alcohol, cleared in xylene, and
embedded in paraffin. Tissue sections from paraffin-embedded tissue
blocks were placed on charged slides, deparaffinized in xylene, rehydrated
through graded alcohol solutions, and stained with H&E or analyzed by
immunohistochemistry.
Patient Samples. Human well-differentiated liposarcoma, dedifferentiated
liposarcoma, lipoma, and normal fat specimens were removed at surgery and
collected from patients treated at Brigham and Women’s Hospital, who gave
informed consent for use of anonymized surgical specimens for research
purposes after all clinically relevant evaluations were performed, with approval of the Partners Health Care Institutional Review Board. The diagnosis
of well-differentiated liposarcoma/atypical lipomatous tumor, dedifferentiated liposarcoma, or lipoma was made by institutional pathologists and
reviewed by E.L.S. and C.D.M.F. to ensure diagnostic accuracy based on criteria of the World Health Organization (2).
Immunohistochemistry. Immunohistochemistry on human samples was performed on a tissue microarray containing three 0.4-mm cores from each
individual tumor, and on selected whole tumor sections. Zebrafish immunohistochemistry was performed on slides of whole zebrafish sections. Slides
were deparaffinized and pretreated with 10 mM citrate (pH 6.0) in a steam
pressure cooker (Decloaking Chamber, BioCare Medical) according to the
manufacturer’s instructions, followed by washing in distilled water. Slides
were pretreated with Peroxidase Block (Dako) for 5 min, followed by serumfree protein block (Dako) for 20 min. Primary rabbit antibody to Ser473
phospho-AKT (#4058; Cell Signaling Technology) or to Ser235/236 phosphoS6 ribosomal protein (#4858; Cell Signaling Technology) was applied at
a 1:50 dilution in Antibody Diluent (Dako) and incubated at 4 °C overnight
(phospho-AKT) or at room temperature for 1 h (phospho-S6). Anti-rabbit
horseradish peroxidase-labeled polymer (Dako) was applied for 30 min.
Immunoperoxidase staining was performed with diaminobenzidine (DAB)+
chromogen kit (Dako), according to the manufacturer’s instructions.
Immunohistochemistry was independently scored by two pathologists
(E.L.S. and S.S.), who then reviewed the few discordant cases together
to arrive at a consensus score. Tumors were scored as positive if >10% of
sarcoma cells exhibited evidence of specific staining for phospho-AKT or
phospho-S6. Positive tumors were further subclassified based on intensity of
staining into either high (strong staining) or intermediate (weak-to-moderate
staining) categories. Normal adipose tissue was used as a negative control.
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Cell Viability Assays. Liposarcoma cells were plated in 96-well plates at 2,000
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cytometry (FACScan; Becton Dickinson) with CellQuest and ModFIT LT software (Becton Dickinson).
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categorical data were assessed via Fisher’s exact test.
ACKNOWLEDGMENTS. We thank G. Molind and L. Zhang for zebrafish
husbandry, J. Testa for the myristoylated mouse Akt2 transgene used in
these studies, and D. E. Fisher for the SU-CCS-1 cell line. This work was
conducted with support from Harvard Catalyst j The Harvard Clinical and
Translational Science Center (National Institutes of Health Award UL1 RR
025758 and financial contributions from Harvard University and its affiliated
academic health care centers), as well as the Ludwig Center at Dana-Farber/
Harvard Cancer Center and Harvard Medical School; A.G. is supported by
National Institutes of Health Grant 1K08CA133103 and is a scholar of the
American Society of Hematology-Amos Medical Faculty Development program; and A.M.-E. is supported by Fundacion Alfonso Martin Escudero.
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